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© Copyright 1996 by the American Chemical Society and the American Society of Pharmacognosy
Volume 59, Number 12
December 1996
Full Papers Novel Cage Indoles from a Malaysian Kopsia Toh-Seok Kam,* K. Yoganathan, and Chen Wei Department of Chemistry, University of Malaya, 59100 Kuala Lumpur, Malaysia Received July 1, 1996X
The EtOH extract of the leaves of Kopsia teoi yielded the octacyclic alkaloids kopsinitarines A-D (1-4) and mersingines A and B (5 and 6), all possessing a novel cage carbon skeleton. The structures of the alkaloids were established by spectral methods and, in the case of kopsinitarine D (4), confirmed by X-ray analysis. The genus Kopsia (Apocynaceae) comprises some 30 species of shrubs and trees distributed mainly over Southeast Asia, India and China.1,2 There are about 12 species distributed over the lowland forest of Peninsular Malaysia and another six in North Borneo.1,2 Some medicinal uses have been reported for the cultivated species K. officinalis, which is used in China for the treatment of rheumatoid arthritis, dropsy, and tonsilitis,2 and in Malaysia the roots of several Kopsia species are used for poulticing ulcerated noses in tertiary syphilis.3 The genus has yielded a prodigious array of new natural products with novel carbon skeletons as well as useful bioactivities.4-14 We have previously reported in preliminary form the occurrence of caged indoles from the leaf extract of Kopsia teoi L. Allorge5,6 and would like now to furnish full details as well as the further isolation of a new member of this class of alkaloids, viz., kopsinitarine D for which we have also carried out X-ray analysis. Results and Discussion The cage alkaloids, kopsinitarines A-D, (1-4), are found in minute amounts (with the exception of kopsinitarine D, 4) only from the leaf extract, while the stem extract provided kopsingine as well as several other aspidofractinine-type alkaloids.7 Two successive extractions of the alkaloids were carried out. In the first study, kopsinitarines A, B, and C (1-3) and mersingines A and B (5, 6) were isolated. In contrast, in the second investigation, involving a different collection of plant * To whom correspondence should be addressed. Tel.: 603-7594266. Fax: 603-7594193. E-mail:
[email protected]. X Abstract published in Advance ACS Abstracts, November 15, 1996.
S0163-3864(96)00538-1 CCC: $12.00
material, kopsinitarines A (1), B (2), and D (4) and mersingine A (5) were isolated. The structures of kopsinitarines A-C have been discussed previously, whereas kopsinitarine D (4) represents a new addition to this group of indole alkaloids. We have previously proposed5 that the kopsinitarines possess an unprecedented cage structure in which an oxygen bridge connects carbons 5 and 17 of the aspidofractinine-like unit, whereas carbons 6 and 16 are joined by a ketonic carbonyl reminiscent of that in kopsin. These structural features give rise to a novel skeletal framework in which
© 1996 American Chemical Society and American Society of Pharmacognosy
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Journal of Natural Products, 1996, Vol. 59, No. 12
Kam et al.
Figure 1. Perspective diagram of kopsinitarine D (4).
Scheme 1
Figure 2. Packing diagram of 4 viewed down the a-axis showing intra- and intermolecular hydrogen bonding.
a central cage unit is formed that is circumscribed by two five-membered rings and three six-membered rings. The isolation of 4 in sufficient quantities has now enabled us to revise some of our previous assignments of the 13C NMR spectra of these compounds (Table 2) and also allowed us to confirm our earlier proposal of the structure by carrying out X-ray analysis. The EIMS of (4) showed a molecular ion at m/z 440 (C23H24N2O7) with mass spectral fragmentation similar to those of compounds 1-3. The UV spectrum showed absorptions at 217, 246, and 288 nm, typical of a dihydroindole chromophore. The 1H and 13C NMR spectral data of 4 showed signals due to a 12-methoxysubstituted dihydroindole, a 16-carbomethoxy group, a 16-OH function, a urethane group on N-1, a ketonic carbonyl (C-22), and three oxymethines (C-5, C-17 and C-15). In common with the other kopsinitarines, the 1H spectrum displayed the characteristic pair of AX doublets at δ 5.25 and 2.79 (J ) 4.9 Hz) due to H-5 and H-6, respectively, as well as the presence of an OH group on C-15. The spectral data thus show that 4 has the same cagelike carbon framework as compounds 1, 2 and 3, with a carbamate substituent on the indole nitrogen and a hydroxyl group on C-15. The stereochemistry of the 15-OH could not be readily established from NMR since the H-15 and 17 resonances are overlapped in 4 and in close proximity in 3,5 precluding NOE experiments. The matter is now resolved unequivocally by an X-ray analysis on 4, allowing confirmation of the structure of the kopsinitarines as well as establishment of the configuration at carbon-15 in the case of 4.
The crystals of 4 are monoclinic belonging to the space group P21, with a ) 8.981(2) Å, b ) 8.724(2) Å, c ) 13.008(1) Å, R ) γ ) 90°, β ) 100.67(1)°, V ) 1001.6(3) Å3; Dx ) 1.460 Mg m-3, and Z ) 2. The structure was solved by the direct method SHELXS8615 and refined by full-matrix least-squares methods. The final R-factor was 0.0702. Both intra- and intermolecular hydrogen bonding were observed, in the former case between O6 (carbamate carbonyl) and 16-OH with the O6 to H distance at 1.963 Å and the O6 to O1 distance at 2.686 Å. The intermolecular hydrogen bond is from O2 (ketonic carbonyl) of one molecule to 15-OH of another at 2.171 Å with the O2 to O4 distance at 2.914 Å. The perspective diagram (Figure 1) confirms the structure deduced from spectral data. In addition to the cage kopsinitarines, we have also isolated trace amounts of compounds 5 and 6. These compounds had NMR spectral data that indicated their structural affinity to the kopsinitarines. In addition, they were shown by HREIMS to possess an additional nitrogen atom. Compounds 5 and 6 gave positive tests with ninhydrin indicating the presence of a primary amino group.16 Furthermore, 5 was readily acetylated (Ac2O/pyridine) to the amide derivative 7. In the case of compound 6, the stereochemistry of the 15-OH could be readily determined, since unlike kopsinitarine C (3), the H-15 and -17 resonances were sufficiently separated. Thus, irradiation of the H-15 signal resulted in NOE enhancement of H-17, indicating that the 15-OH is R. We believe that compounds 5 and 6 are probably artifacts derived from further reaction of the kopsinitarines in the basic media under which extraction of alkaloids was carried out. One possible genesis of these
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Cage Indoles from a Malaysian Kopsia Table 1. H 3 5 6 9 10 11 14 15 17 18 19 21 16-OH ArOMe NCO2Me a
1H
Journal of Natural Products, 1996, Vol. 59, No. 12 1111
NMR Spectral Data for Compounds 1-6 (270 MHz, CDCl3)a 1
2
3
4
5
6
3.67 m 3.76 m 5.20 d (4.9) 2.78 d (4.9) 6.90 dd (7.3, 1) 7.12 dd (7.8, 7.3) 6.86 dd (7.8, 1) 6.07 dt (10, 2.5)
3.68 m 3.78 m 5.16 d (4.9) 2.73 d (4.9) 6.87 dd (7.3, 1) 6.79 dd (7.8, 7.3) 6.71 dd (7.8, 1) 6.05 dt (10, 2.5) 5.66 dt (10, 2) 3.75 m 1.63 m 1.78 m 1.63 m 1.63 m 3.53 d (2)
5.59 dt (10, 2) 3.46 d (2) 1.67 m 1.73 m 1.52 m 1.63 m 3.54 d (2)
3.25 dd (14, 6.4) 3.51 td (14, 4.9) 5.18 d (4.9) 2.68 d (4.9) 6.87 dd (7.3, 1) 6.78 dd (7.8, 7.3) 6.68 dd (7.8, 1) 1.56 m 2.71 m 3.87 m 3.63 d (2) 1.69 m 2.08 m 1.03 m 1.69 m 4.05 d (2)
3.80 s
3.79 s
3.25 dd (14, 6.4) 3.50 td (14, 4.9) 5.25 d (4.9) 2.79 d (4.9) 6.92 dd (7.3, 1) 7.11 dd (7.8, 7.3) 6.85 dd (7.8, 1) 1.57 m 2.70 m 3.93 m 3.93 m 1.64 ddd (14, 11, 8.5) 2.18 ddd (14, 11, 2) 1.19 ddd (14, 11, 8.5) 2.04 ddd (14, 11, 2) 3.93 m 6.67 s 3.82 s 3.78 s
3.68 m 3.77 m 5.14 d (4.9) 2.67 d (4.9) 6.87 dd (7.3, 1) 6.79 dd (7.8, 7.3) 6.69 dd (7.8, 1) 6.07 dt (10, 2.5)
5.60 dt (10, 2) 3.40 d (2) 1.61 m 2.23 m 1.53 m 1.66 m 3.65 d (2) 6.70 s 3.83 s 3.77 s
3.25 dd (14, 6.4) 3.52 td (14, 4.9) 5.20 d (4.9) 2.73 d (4.9) 6.87 dd (7.3, 1) 6.78 dd (7.8, 7.3) 6.68 dd (7.8 1) 1.57 m 2.71 m 3.91 m 3.87 d (2) 1.68 m 2.09 m 1.08 m 1.68 m 4.04 d (2)
3.79 s
3.78 s
Assignments based on COSY and COSYLR.
compounds derives from initial formation of an isokopsin-like precursor 8 formed under basic conditions via a reversible acyloin rearrangement17 of the kopsinitarine precursor (2 or 3). This could be followed by a reversible condensation with ammonia resulting in the unstable imine 9, which could then subsequently rearrange back to the original kopsin-like alkaloid (5 or 6), which now incorporates a 16-R-amino function as shown in Scheme 1. Unfortunately, we could not verify this proposal due to paucity of material in the case of compounds 2 and 3, and in the case of kopsinitarine D (4), attempted reaction failed to yield the corresponding 16-R-amino derivative. One possible explanation for this is that in the case of 4 (as well as in 1) where a carbamate function is present, the 16-OH is unreactive due to H-bonding involving the promimate carbamate carbonyl function. This proposal is supported by the observation that only mersingines (e.g., 5 and 6) derived from kopsinitarine partners in which the indole nitrogen does not carry a urethane substituent (e.g., 2 and 3) were isolated, whereas the corresponding artifacts derived from kopsinitarines A and D (1 and 4) were notably absent. This explanation is also supported by the observation of such intramolecular H-bonding even in the solid state (Figure 2). Experimental Section General Experimental Procedures. All melting points were uncorrected. UV spectra were recorded on a Shimadzu UV-160A spectrophotometer. Optical rotations were determined on a JASCO DIP-370 digital polarimeter. Mass spectra were obtained on a VG ProSpec spectrometer. 1H and 13C NMR spectra were recorded in CDCl3 using TMS as internal standard on a JEOL JNM-GSX 270 spectrometer at 270 and 67.8 MHz, respectively. Plant Material. Plant material was collected in Johore, Malaysia. Voucher specimens (3976) are deposited at the Herbarium, Department of Chemistry, University of Malaya. Extraction and Isolation. Extraction of the ground leaves was carried out in the usual manner by partitioning the concentrated EtOH extract with dilute acid as has been described in detail elsewhere.7,10 The alkaloids were isolated by initial column chromatography on Si gel using CHCl3 with increasing proportions of MeOH followed by rechromatography of appropriate partially resolved fractions using centrifugal TLC.
Table 2. 13C NMR Spectral Data for Compounds 1-6 (67.8 MHz, CDCl3)a C
1
2
3
4
5
6
2 3 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 ArOMe NCO2Me NCO2Me CO
72.2 47.4 95.8 56.2 59.4 136.6 113.2 126.8 115.0 149.7 131.0 131.0 128.6 87.5 87.8 18.6 25.6 31.1 62.2 56.1 53.5 156.2 205.6
68.1 47.5 95.9 56.6 59.7 131.0 115.2 120.6 110.5 145.9 139.8 130.8 129.1 86.3 87.9 19.4 26.2 32.3 62.1 55.3
68.3 42.2 95.2 58.3 60.0 131.3 115.4 120.6 110.6 145.9 139.3 32.6 70.8 87.1 88.7 19.4 23.2 35.9 60.3 55.4
68.8 47.4 95.9 57.4 60.0 131.6 115.3 120.6 110.4 145.9 139.8 130.7 129.1 72.0 87.7 20.0 26.1 32.0 62.3 55.3
69.0 42.1 95.3 59.0 60.3 131.8 115.4 120.7 110.4 146.6 139.7 32.6 70.9 72.6 88.5 20.1 23.1 35.6 60.5 55.3
207.0
206.9
72.2 42.1 95.1 57.5 59.5 136.6 113.0 126.8 115.1 149.5 130.3 32.4 70.3 88.1 88.3 18.5 22.2 34.5 60.2 56.0 53.5 156.1 205.9
209.0
208.9
a
Assignments based on HMQC and HMBC.
Solvent systems used for centrifugal TLC were Et2O, Et2O-EtOAc (3:1), Et2O-EtOAc (8:1), and Et2O-hexane (2:1). The yields (g kg-1) of the alkaloids 1-6 are as follows: Extraction 1: 1 (0.004), 2 (0.003), 3 (0.003), 5 (0.007), 6 (0.003). Extraction 2: 1 (0.002), 2 (0.001), 4 (0.019), 5 (0.002). Kopsinitarine A (1): [R]D ) +23° (CHCl3, c 0.093), UV (EtOH) λmax (log ) 215 (4.41), 245 (3.85), 289 (3.06); EIMS m/z (rel int) 422 [M+] (100), 394 (31), 393 (63), 363 (14), 362 (15), 335 (22) 307 (5.5); HREIMS, M+ found 422.145, calcd for C23H22N2O6 422.147; 1H NMR and 13C NMR, see Tables 1 and 2. Kopsinitarine B (2): [R]D ) +188o (CHCl3, c 0.051); UV (EtOH) λmax (log ) 212 (4.60), 246 (3.90), 290 (3.33); EIMS m/z (rel int) 364 [M+] (100), 335 (52), 307 (7); HREIMS M+ found 364.1433, calcd for C21H20N2O4 364.1423 1H NMR and 13C NMR, see Tables 1 and 2. Kopsinitarine C (3): [R]D ) -96o (CHCl3, c 0.026); UV (EtOH) λmax (log ) 213 (4.49), 246 (3.80), 290 (3.15); EIMS m/z (rel int) 382 [M+] (90), 354 (52), 353 (55), 337 (15), 309 (12); HREIMS M+ found 382.1529, calcd for C21H22N2O5 382.1529; 1H NMR and 13C NMR, see Tables 1 and 2.
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Table 3. Atomic Coordinates (×104) and Equivalent Isotropic Displacement Parameters (A2 × 103) for 4a atom
x
y
z
U (eq)
O(1) O(2) O(3) O(4) O(5) O(6) O(7) N(1) N(4) C(2) C(3) C(5) C(6) C(7) C(8) C(9) C(10) C(11) C(12) C(13) C(14) C(15) C(16) C(17) C(18) C(19) C(20) C(21) C(22) C(23) C(24) C(25)
-4004(3) -3000(4) -696(3) -965(4) -6948(3) -6556(4) -8405(3) -5736(3) -747(3) -4461(4) 607(4) -790(4) -2380(4) -3347(4) -4400(4) -4208(5) -5453(6) -6848(5) -7059(4) -5786(4) 728(5) -762(5) -3441(4) -1959(4) -4975(4) -3600(5) -2118(4) -2181(4) -2947(4) -6434(4) -7672(7) -9728(5)
-3039(4) -435(4) -3289(3) -8115(3) -713(4) -1812(4) -2786(4) -2953(4) -4355(4) -3888(4) -5319(6) -3003(5) -2329(4) -3826(4) -3715(4) -4130(5) -4009(5) -3551(5) -3145(5) -3185(4) -6075(6) -6774(5) -3176(5) -4210(5) -5503(4) -6460(4) -5678(4) -5128(4) -1744(5) -1813(5) 552(6) -3042(11)
-194(2) -1315(3) -1043(2) -1886(2) 2874(2) -1287(2) -4266(2) -2650(2) -2750(2) -2087(3) -2437(3) -2142(3) -2557(3) -2877(3) -3909(3) -4899(3) -5714(3) -5537(3) -4539(3) -3738(3) -1368(3) -1231(3) -1117(3) -880(3) -1885(3) -1417(3) -1544(3) -2667(3) -1611(3) -2201(3) -2441(4) -5014(5)
43(1) 52(1) 36(1) 44(1) 42(1) 50(1) 50(1) 30(1) 31(1) 24(1) 41(1) 35(1) 31(1) 24(1) 27(1) 35(1) 42(1) 41(1) 34(1) 28(1) 42(1) 35(1) 29(1) 30(1) 32(1) 34(1) 26(1) 25(1) 33(1) 31(1) 62(2) 81(2)
a U(eq) is defined as one third of the trace of the orthogonalized Uij tensor.
Kopsinitarine D (4): colorless crystals (CH2Cl2-ether); mp 258-260 oC; [R]D ) -46o (CHCl3, c 0.079); UV (EtOH) λmax (log ) 217 (4.43), 246 (3.86), 288 (3.03); EIMS m/z (rel int) 440 [M+] (100), 422 (65), 412 (20), 411 (70), 394 (10), 393 (50), 380 (18), 363 (5), 353 (15), 335 (15), 123 (5), and 109 (5); 1H NMR and 13C NMR see Tables 1 and 2. Mersingine A (5): [R]D ) +88o (CHCl3, c 0.058); UV (EtOH) λmax (log e) 213 (4.54), 247 (3.85), 290 (3.28); EIMS m/z (rel int) 363 [M+] (90), 335 (50), 334 (100), 307 (15); HREIMS, M+ found 363.1578, calcd for C21H21N3O3 363.1583; 1H NMR and 13C NMR, see Tables 1 and 2. Mersingine B (6): [R]D ) -82o (CHCl3, c 0.022); UV (EtOH) λmax (log e) 213 (4.72), 246 (4.05), 290 (3.52); EIMS m/z (rel int) 381 [M+] (100), 353 (94), 352 (95), 336 (24), 335 (24), 334 (34); HREIMS M+ found 381.1685, calcd for C21H23N3O4 381.1688; 1H NMR and 13C NMR, see Tables 1 and 2. Acetylation of 5 to 7. Ac2O (0.02 mmol) was added to a stirred solution of (5) (0.04 mmol) in pyridine (1.5 mL) at 27 °C. After ca. 2.5 h, saturated Na2CO3 (5 mL) was added, and the mixture was extracted with CHCl3 (2 × 10 mL). The extract was then dried (Na2SO4) and the solvent evaporated. Flash chromatograpthy over SiO2 (1% MeOH-CHCl3) afforded 4.8 mg (81%) of (7): EIMS m/z (rel int):405 [M+] (100), 376 (48), 364 (15), 335 (16), 334 (15), 305 (25); HREIMS M+ found 405.1686, calcd for C23H23N3O4 405.1688; UV (EtOH) λmax (log e) 213 (4.51), 247 (3.86), 289 (3.28); 1H NMR (CDCl3, 270 MHz) δ 1.45 (ddd, J ) 13.3, 11.3, 2.3 Hz, H-19), 1.77 (ddd, J ) 13.3, 11.3, 7.3 Hz, H-18), 1.88 (ddd, J ) 13.3, 11.3, 2.3 Hz, H-18), 2.10 (s, NHCOMe), 2.40 (ddd, J ) 13.3, 11.3, 7.3 Hz, H-19), 2,79 (d, J ) 4.9 Hz, H-6), 3.50 (d, 2 Hz, H-21), 3.70-3.84 (m, 2 × H-3), 3.79 (s,
Kam et al.
12-OMe), 4.45 (d, J ) 2 Hz, H-17), 5.19 (d, J ) 4.9 Hz, H-5), 5.71 (dt, J ) 10, 2 Hz, H-15), 6.06 (dt, J ) 10, 2.5 Hz, H-14), 6.71 (dd, J ) 7.8, 1 Hz, H-11), 6.80 (dd, J ) 7.8, 7.3 Hz, H-10), 6.88 (dd, J ) 7.3, 1 Hz, H-9); 13C NMR (CDCl3, 67.8 MHz) δ 21.9 (C-19), 24.2 (NHCOMe) 26.4 (C-18), 32.9 (C-20), 47.4 (C-3), 55.4 (12-OMe), 57.4 (C6), 61.0 (C-7), 63.2 (C-21), 70.7 (C-2), 72.5 (C-16), 83.1 (C-17), 96.5 (C-5), 110.4 (C-11), 115.4 (C-9), 120.9 (C10), 129.5 (C-15), 130.5 (C-14), 131.3 (C-8), 139.5 (C13), 146.1 (C-12), 170.3 (NHCOMe), 204.2 (C-22). X-ray Diffraction Analysis. A total of 3762 reflections were collected up to θmax of 25° on a CAD4 diffractometer at 27 °C using Mo KR (λ ) 0.710 73 Å). The data were collected by the ω-2θ method, 3523 observed reflections with I > 2σ(I), and were corrected for Lorentz-polarization effect but not for absorption. The structure was solved by using the direct method SHELXS86.15 All non-hydrogen atoms were refined anisotropically by full-matrix least-squares refinement on an IBM 486 PC to R ) 0.0702, wR ) 0.1719 for the observed reflections, w ) [σ2(Fo2) + (0.1293P)2 + 0.2044P]-1 where P ) (Fo2 + 2Fc2)/3. Hydrogen atoms were generated geometrically at C-H 0.95 Å and were allowed to ride on their respective parent atoms with B fixed at 1.3 times that of the parent atom. The atomic coordinates for the non-hydrogen atoms and their equivalent isotropic displacement parameters are given in Table 3. Calculated coordinates for the hydrogen atoms, anisotropic displacement parameters for the nonhydrogen atoms, a full list of bond distances and angles, and the structure factor table have been deposited at the Cambridge Crystallographic Data Center.18 Acknowledgment. We thank the University of Malaya and IRPA for financial support and Dr. J. K. MacLeod, Research School of Chemistry, Australian National University, for mass spectra of compounds 3 and 6. References and Notes (1) Markgraf, F. Blumea 1972, 20, 416-425. (2) Sevenet, T.; Allorge, L.; David, B.; Awang, K.; Hadi, A. H. A.; Kan-Fan, C.; Quirion, J. C.; Remy, F.; Schaller, H.; Teo, L. E. J. Ethnopharmacol. 1994, 41, 147-183. (3) Burkhill, I. H. A Dictionary of the Economic Products of the Malay Peninsula; Ministry of Agriculture and Cooperatives: Kuala Lumpur, Malaysia, 1966. (4) Kam, T. S.; Yoganathan, K.; Chuah, C. H. Tetrahedron Lett. 1993, 34, 1819-1822. (5) Kam, T. S.; Yoganathan, K.; Chuah, C. H. Tetrahedron Lett. 1994, 35, 4457-4460. (6) Yoganathan, K.; Wong, W. H.; Kam, T. S. Nat. Prod. Lett. 1995, 5, 309-314. (7) Kam, T. S.; Yoganathan, K.; Chuah, C. H.; Chen, W. Phytochemistry 1993, 32, 1343-1346. (8) Kam, T. S.; Yoganathan K.; Chuah C. H. Tetrahedron Lett. 1995, 36, 759-762. (9) Kam, T. S.; Tan, P. S.; Hoong, P. Y.; Chuah, C. H. Phytochemistry 1993, 32, 489-491. (10) Kam, T. S.; Tan, P. S. Phytochemistry 1990, 29, 2321-2322. (11) Homberger, K.; Hesse, M. Helv. Chim. Acta 1982, 65, 25482557. (12) Homberger, K.; Hesse, M. Helv. Chim. Acta 1984, 67, 237-248. (13) Ruangrungsi, N.; Likhitwitayawuid, K.; Jongbunprasert, V.; Ponglux, D.; Aimi, N.; Ogata, K.; Yasuoka, M.; Haginiwa, J.; Sakai, S. Tetrahedron Lett. 1987, 28, 3679-3682. (14) Awang, K.; Sevenet, T.; Pais, M.; Hadi, H. J. Nat. Prod. 1993, 56, 1134-1139. (15) Sheldrick, G. M. Acta Crystallogr. 1990, A46, 467-473. (16) Kobayashi, J.; Cheng, J.-F.; Ohta, T.; Nozoe, S.; Ohizumi, Y.; Sasaki, T. J. Org. Chem. 1990, 55, 3666-3670. (17) Govindachari, T. R.; Nagarajan, K.; Schmid, H. Helv. Chim. Acta 1963, 45, 433-444. (18) The coordinates can be obtained, upon request, from Dr. Olga Kennard, University Chemical Laboratory, 12 Union Road, Cambridge CB2 1EZ, U.K.
NP960538B
